Flat plate electrophoresis

ABSTRACT

A device for horizontal flat plate electrophoresis comprising in combination a horizontal plate defining a rectangular gel bed, surrounded by a ledge, the height of which defines the height of the gel, and by lateral walls, a cover adapted to rest on the ledge, fixing the thickness of the gel, two legs attached to the bottom of the plate and extennding along the entire width of the plate at each end, a slot being provided at each end of the gel bed and running the width of same said slots extending through the entire height of the leg, the cover being provided with a plurality of holes for the insertion of templates during the polymerization of the gel, and for the subsequent insertion of the samples. The gel bed can be divided into a plurality of separate channels, and openings for the insertion of samples are provided near the edge of the cover or at about its middle.

[ June 10, 1975 FLAT PLATE ELECTROPI-IORESIS Primary ExaminerJ0hn I-I.Mack Assistant Examiner-A. C. Prescott Attorney, Agent, orFirmOstrolenk, Faber, Gerb & Soffen [75] Inventors: David Elson; ShlomoAvital, both of Rehovot, Israel [73] Assignee: Yeda Research &Development Co.,

[57] ABSTRACT A device for horizontal flat plate electrophoresis com-Ltd., Rehovot, Israel May 25, 1973 [22] Filed:

prising in combination a horizontal plate defining a [21] Appl' 363961rectangular gel bed, surrounded by a ledge, the height of which definesthe height of the gel, and by lateral walls, a cover adapted to rest onthe ledge, fixing the thickness of the gel, two legs attached to thebottom of the plate and extennding along the entire width of 09 m w s kNEW 9 9 P ,0 4 mm 0 u/ 2 "4 .00 mm mmh "6 .r ""3 "U .L he... '0 d Std iU.mF 1]] 2 00 555 III the plate at each end, a slot being provided ateach [56] References Cited UNITED STATES PATENTS end of the gel bed andrunning the width of same said slots extending through the entire heightof the leg,

204/299 X the cover being provided with a plurality of holes for theinsertion of templates during the polymerization of 204/299 the gel, andfor the subsequent insertion of the sam- 204/299 ples. The gel bed canbe divided into a plurality of separate channels, and openings for theinsertion of G O 00 H 4 O 2 3,482,943 12/1969 Csizmas et a].

3,635,808 l/l972 Elevitch..i..........

3,674,678 7/1972 Post, Jr. et al.....

3,773,646 11/1973 Mandle et OTHER PUBLICATIONS Nanto et a1, OptimalCond. in Starch-Gel Elecphor.

of Heat Denatured Collagen, Jrnl. Amer. Leather samples are providednear the edge of the cover or at about its middle.

Chemists Assoc., Vol. LX, No. 2, Feb. 1965, pp. 3 71 7 Claims, 2 DrawingFigures SHEET PATENTEDJUH 10 I975 FLAT PLATE ELECTROPHORESIS BACKGROUNDOF THE INVENTION 1. Field of the Invention The invention relates to anovel device for horizontal flat plate electrophoresis; it can be usedfor onedimensional and for two-dimensional runs and is usedadvantageously with polyacrylamide gel electrophoresis.

2. Description of the Prior Art A number of instruments have beendescribed for electrophoresis in flat gel slabs. Both vertical andhorizontal arrangements have been employed, each having its ownadvantages and disadvantages. The vertical arrangement is amenable tothe use of discontinuous gel and buffer systems; different slot-formingtemplates can be used interchangeably to vary the number and size ofsample wells; sample loading is easily accomplished without opening theplates that enclose the gel; and the ends of the gel are in directcontact with the electrode buffers, obviating the need for filter paperor other connecting bridges. The horizontal instruments described lackthese features but, having no upper buffer chamber, are simpler toconstruct and having the additional advantage that samples may beapplied in the center of the gel as easily as at the end.

According to the invention, there is provided a horizontal apparatusthat incorporates a number of the advantages of both types ofinstrument. The ends of the gel, bent at right angles to the horizontalbody, make direct contact with the electrode buffers, so that neither aconnecting bridge nor a water-tight upper buffer chamber is needed.Interchangeable plates enable sampels to be placed either at the end ofthe gel or in the center. Interchangeable removable templates afford awide range of sample volumes and allow the sample to be applied withoutopening the instrument. The apparatus can be used for oneandtwo-dimensional electrophoresis and, in certain cases, two parallelsamples can be run in the same two-dimensional gel, facilitatingcomparison. Its use is illustrated with ribosomal prote- DESCRIPTION OFTHE PRESENT INVENTION The present invention relates to a novel devicefor horizontal flat plate electrophoresis.- The novel device can be usedfor both oneand two-dimensional runs. It is advantageously used withpolyacrylamide gel electrophoresis, which has become one of the mostwidely used techniques for the analytical separation of biologicalmacromolecules.

The novel device according to the present invention is described by wayof example only with reference to the following detailed description andwith reference to the enclosed drawings, in which:

FIG. 1 is a schematical exploded view of a device according to theinvention, illustrating a number of different embodiments;

FIG. 2 is a schematical exploded view of another device according to theinvention.

The device according to the invention is designed for horizontal flatplate gel electrophoresis in one or two dimensions. It consistsessentially of a box and a cover, the space between these defining thegel volume. In the following, there are set out two versions, Version Adefining a continuous rectangular gel bed; Version B defining a gel beddivided into a plurality of individual elongated gel strips.

The following description contains by way of example definitedimensions. These relate to certain laboratory models made and tested.The dimensions are clearly illustrative and not to be construed in anylimitative manner.

Version A is shown in FIG. 1. The box comprises a perspex plate l1, l7cm square and 1.2 cm thick, into which is machined a 14 cm X 14 cmsquare gel bed 12, and a surrounding ledge 13, 1 cm wide and 2 mm higherthan the bed. A cover 14 rests on the ledge, fixing the thickness of thegel at 2 mm. Two perspex legs 15 1.8 cm wide and L6 cm high arepermanently fixed at right angles to the bottom of the plate and theseextend the entire width of the plate of 17 cm, one at each end. At eachend of the gel bed 12 there is a slot 16 2 mm wide running the width ofthe gel bed, and extending downward through the entire height of theleg, which is open at its bottom.

In use, the slots 16 are closed off at the bottom of the legs with arubber strip 17 supported by a perspex strip 18 held in place by rubberbands stretched back and forth across it and anchored to lugs 19 on bothsides of the leg. A cover, 14 (see below) is placed on the ledge 13,completely covering the gel bed, and is clamped in place with springclips, which are not shown. Templates 20 are inserted into the slots 21in the cover and are pressed down to touch the bottom of the gel bed(see below). The gel bed and leg slots are filled with gel mixturethrough one of the slots 21 in the cover, through which air alsoescapes. The apparatus now contains a continuous gel 2 mm thick thatextends from the bottom of one leg through the gel bed to the bottom ofthe other leg. After the gel has polymerized, the templates are removed,leaving holes in the gel for the samples. (In case of a pre-run withoutsample, the templates are left in place.) The perspex and rubber stripsare removed from the legs, exposing the two ends of the gel. Theapparatus is placed on two rectangular electrode vessels filled withbuffer, with the plate resting horizontally on the opposing rims of thetwo vessels and each leg dippling down into the buffer of a differentvessel so that the gel is in contact with the buffer. The samples arepipetted through the template slots into the holes left by thetemplates, and the electrophoretic run is made.

At the end of the run the cover is gently disengaged from the gel byremoving the clips and by turning the screws 22 which press on the ledge13. This allows the cover to be raised slowly in a controlled manner andprevents the gel from tearing. The gel should be lubricated during thisoperation by introducing water or another suitable liquid through theslot 21. After removal of the cover, a knife or spatula is moved aroundthe periphery of the gel, and the lubricated gel is cut loose from itsextensions into the legs, gently disengaged from the gel bed with theaid of a spatula, and lifted out.

A second version (B) of the apparatus is shown in FIG. 2. It resemblesversion A except that the gel bed is divided into eleven separatechannels 23, and 23 each 14 cm long, 2 mm deep and either 1.0 or 0.6 cmwide, respectively. The slots leading from the gel bed through the legsare also separate.

Three types of cover 14 are used with version A (FIG. 1). Type 14 isemployed only from twodimensional runs, described below. Types 14 and 14are used for both analytical and preparative runs, and are identicalexcept that the samples are placed near one end of the gel with 14 andin the center of the gel with 14. For analytical runs, a templatecarrier 24 is fixed in the carrier slot 25. Template carrier 24;contains l4 template slots 26, each of 6 X 2 mm. The templates 20 madeof, e.g., polyethylene or teflon, have a rectangular cross section of 6X 2 mm to fit the slots 26. The last protruding 2 mm of template 20,i.e., the part that enters the gel, is shaped in rectangles of differentsize, 2, 3, 4 or 6 mm long and either 1 mm wide (at one end of thetemplate) or 2 mm wide (at the other end). For a gel 2 mm thick, thecorresponding sample volumes range from 4 to 24 ,ul. Fourteen samples ofdefined volume can thus be run in a single gel. Another template 20 withtwo protrusions at either end, forms two sample holes, each 2 X 2 mmallowing up to 28 samples to be run simultaneously. Identical ordifferent templates may be used. Unused template slots are plugged witha 6 X 2 mm template inserted so that its flat end is flush with theunder surface of the cover and does not enter the gel. For perparativeruns, the template carrier 24 is replaced by a single perspex template24 defining a sample bed 2 mm wide and 12 cm long, accommodating asample of 0.5 ml in a gel of 2 mm thickness. In some cases it isfeasible to increase the template width to as much as 6 mm, raising thesample volume to 1.5 ml.

The covers 14 of instrument B, shown in FIG. 2, are identical withcovers 14 and 14 of A, except that the template slots are cut directlyinto the cover 14.

In both A and B, number 30 from 1 to 14 and from 1 to 11, respectively,are engraved into the underside of the cover and become embossed on thegels. When the gels are removed, the numbers make it easy to orient thegel properly, to identify each sample in gel slabs from instrument A,and to identify each gel strip from instrument B. This makes it possibleto process (e.g., stain and destain) a number of strips together in thesame vessel without confusing them. When the sample is placed in thecenter of the gel, the numbers at each end, oriented oppositely withrespect to the center or otherwise marked to distinguish one from theother, identify the anode and cathode ends.

In two-dimensional electrophoresis, the first run is made in one channelof instrument B. (A duplicate sample can be run in a parallel channel]to monitor the run.) The strip is then placed in the gel bed of instrument A parallel to the slots 16, anywhere along the length of the gelbed, provided that it does not cover either of the slots 16. The ends ofthe gel are trimmed so as to leave a small space between each end of thegel and the wall of the gel bed. Cover 14 (FIG. 1) is clamped in placeand the second gel is poured, embedding the first gel in it. A run iscarried out and after the run, the gel is removed as described above.

When the two-dimensional pattern is suitable, it is possible to run twosamples in the same gel, facilitating comparison. In the firstdimension, with instrument B and cover 14 one sample is placed in theregular template slot 26 with template 20 or 20 A second template slot,and template 20 are provided for the same gel strip, two or three cmcloser to the center of the gel. The samples may be slightly displacedfrom each other laterally to give, in the first dimension, two separatepatterns lying side by side but displaced from each other longitudinallyor else the two samples may share the same migration path. Botharrangements have given satisfactory results.

The major advantages of the instruments are their simplicity, ease ofuse and versatility. The various covers and templates afford a widerange of sample volumes, both for analytical and preparative purposes.No difficulties are encountered with unusually soft or hard gels.Samples may as easily be placed in the center of the vgel as at the end,using the template slots 26;, of cover 14 allowing the simultaneoushandling of components that migrate in opposite directions. Instrument Ais suitable for the simultaneous analysis of a large number of samplesin a single gel slab. With instrument B, it is possible to pour only asmany gel strips as are needed for a small number of samples, to use gelsof different porosities in different channels (when passing from oneporosity to another, it is well to wait half an hour, to prevent thepossibility of mixing of the different gels), and to run differentchannels for different periods of time (electrophoresis can be stoppedin a single channel by interrupting the run and gouging the gel out ofthe leg slots of the channel to be stopped, after which the run isresumed).

Since the ends of the gel make direct contact with the electrode bufferswhen the plate is horizontal, there is no need for a verticalarrangement or for cloth or filter paper bridges. There are no specialrequirements for the electrode vessels. Ordinary plastic refrigeratorstorage boxes and suitably bent stainless steel rods make satisfactoryvessels and electrodes.

After destaining, it is often useful to dehydrate the gel by placing itin 55-60% aqueous ethanol. This causes the gel to shrink to about aquarter of its original size within about a day, with one change ofsolvent, sharpening and intensifying the stained zones and making thegel stiffer and more resistant to tearing. At higher ethanolconcentrations, the gel also becomes opaque and white, increasing thecontrast between stain and background. The ethanol concentrationrequired for this varies with the nature of the gel and must be foundempirically. For gels of acrylamide and bisacrylamide concentrations of7.5% and 0.2% or 20 and 0.2%, ethanol has been satisfactory. Gels of 18and 0.5% have been damaged by this treatment but have responded well to65 70% ethanol. Dehydrated gels may be removed from the ethanol bath,airdried, and stored for long periods without special precautions.During drying, the corners of the gel should be weighted down to preventcurling. Alternatively, if excess moisture is removed from one side ofthe gel with an air stream, the partially dried side adheres tocardboard and the gel dries flat and remains mounted on the cardboard.Dehydrated gels may be rehydrated in 7% acetic acid, where they regaintheir original size and transparency within an hour or two.

The optimal conditions for a gel-buffer system must be foundempirically. If migration is faster in the middle of the gel than at thesides, the effect can be reduced or eliminated by lowering thetemperature (doing the run in a cold room instead of the laboratory) andby reducing heat production by lowering the voltage (the duration of therun is increased accordingly). It seems best to regulate the voltagerather than the current. Particularly in second-dimensional runs, whereno equilibrating pre-run is possible, the current may drop considerablyduring the run. At constant voltage, this does not significantly affectmigration and progressively reduces heat production. If the current werekept constant, the voltage would rise, increasing heat production andalso increasing the migration rate in a way not always fully controlled.Since there may be an appreciable voltage drop within the buffervessels, the voltage should be adjusted with an external voltmeter whoseelectrodes are placed close to the ends of the gel.

We claim:

l. A device for horizontal flat plate electrophoresis comprising incombination,

a horizontal plate having an upper and a lower surface and surrounded bylateral walls, the upper surface of the plate and the lateral wallsdefining a rectangular gel bed;

a step of predetermined height in each of the lateral walls therebydefining a ledge;

a cover adapted to rest on the ledge and thereby define the thickness ofthe gel bed;

a plurality of holes in the cover for the insertion of templates duringpolymerization of the gel to define predetermined holes of predeterminedvolume in the gel and for insertion of samples of predetermined volumein the holes;

two legs attached to the lower surface of the plate at opposing sidesthereof and coextensive with the sides to which the legs are attached;and

a slot through the plate positioned over each leg, ex-

tending for the full length of the leg, and extending through the entireheight of the leg.

2. The device of claim 1 wherein the horizontal plate defines a squaregel bed.

3. The device of claim 2 wherein the gel bed is divided into a pluralityof separate channels, defining individual gel strips, separate from eachother.

4. The device of claim 2 wherein the holes in the cover for theinsertion of templates are located near one of the edges of the coverand about the middle of the cover.

5. The device of claim 2 further comprising templates of varyingpredetermined volume so as to define in the gel bed space forpredetermined vaying samples of predetermined varying volume.

6. The device of claim 2 wherein the gel bed is divided into a pluralityof separate channels, defining individual gel strips, separate from eachother, and wherein two of the holes in the cover are located above thesame channel, one of which is positioned near one of the slots and theother hole is offset toward the center of the cover.

7. The device of claim 6 further comprising templates in each of the twoholes, each of the templates being of a different shape.

1. A DEVICE FOR HORIZONTAL FLAT PLATE ELECTROPHORESIS COMPRISING INCOMBINATION, A HORIZONTAL PLATE HAVING AN UPPER AND A LOWER SURFACE ANDSURROUNDED BY LATERAL WALLS, THE UPPER SURFACE OF THE PLATE AND THELATERAL WALLS DEFINING A RECTANGULAR GEL BED; A STEP OF PREDETERMINEDHEIGHT IN EACH OF THE LATERAL WALLS THEREBY DEFINING A LEDGE; A COVERADAPTED TO REST ON THE LEDGE AND THEREBY DEFINE THE THICKNESS OF THE GELBED; A PLURALITY OF HOLES IN THE COVER FOR THE INSERTION OF TEMPLATESDURING POLYMERIZATION OF THE GEL TO DEFINE PREDETERMINED HOLES OFPREDETERMINED VOLUME IN THE GEL AND FOR INSERTION OF SAMPLES OFPREDETERMINED VOLUME IN THE HOLES; TWO LEGS ATTACHED TO THE LOWERSURFACE OF THE PLATE AT OPPOSING SIDES THEREOF AND COEXTENSIVE WITH THESIDES TO WHICH THE LEGS ARE ATTACHED; AND A SLOT THROUGH THE PLATEPOSITIONED OVER EACH LEG, EXTENDING FOR THE FULL LENGTH OF THE LEG, ANDEXTENDING THROUGH THE ENTIRE HEIGHT OF THE LEG.
 2. The device of claim 1wherein the horizontal plate defines a square gel bed.
 3. The device ofclaim 2 wherein the gel bed is divided into a plurality of separatechannels, defining individual gel strips, separate from each other. 4.The device of claim 2 wherein the holes in the cover for the insertionof templates are located near one of the edges of the cover and aboutthe middle of the cover.
 5. The device of claim 2 further comprisingtemplates of varying predetermined volume so as to define in the gel bedspace for predetermined vaying samples of predetermined varying volume.6. The device of claim 2 wherein the gel bed is divided into a pluralityof separate channels, defining individual gel strips, separate from eachother, and wherein two of the holes in the cover are located above thesame channel, one of which is positioned near one of the slots and theother hole is offset toward the center of the cover.
 7. The device ofclaim 6 further comprising templates in each of the two holes, each ofthe templates being of a different shape.